The Association Between Notch Pathway And Early Spontaneous Abortion And The Effects Of Notch Pathway On The Survival And Angiogenesis Of Trophoblast Cells

Part one.The expression of Notch-1 related proteins in the villus tissues of patients with early spontaneous abortionBackground and Objective:Early spontaneous abortion(ESA)occurs in the first 12 weeks of gestation,is a common clinical syndrome with the highest incidence rate among all spontaneous abortion cases.Although the pathogenesis of ESA has not yet been completely understood,inadequate invasion and/or abnormal vasculogenesis or angiogenesis are considered as direct causes of ESA.Trophoblast cells from chorionic tissues are the key cells for invasion and angiogenesis during placentation.Clinical researches reported that Notch pathway was downregulated in trophoblast cells of preeclamptic placenta.Targeted disruption or knockout of Notch-1 in mice resulted in embryonic lethality with severe vascular defects in placenta.In vitro studies pointed out that Notch-1 pathway could promote the differentiation,migration and invasion of trophoblast cells.However,the expressions of Notch related proteins in the ESA villus tissues especially in trophoblast cells and their roles in ESA have rarely been reported.The present study aimed to detect the pathological changes and Notch related protein expressions in villus tissues from patients with ESA and evaluate the potential relationships between them.Methods:1.30 cases of villus tissues from ESA patients and another 30 cases of villus tissues from induced abortion females as healthy control were collected.HE staining was employed to observe and compare the pathologic differences between above two groups.2.Real-time PCR(RT-PCR),Western blot and immunohistochemical analysis were used to detect the expressions of VEGF、VEGFR1 and VEGFR2 in villus tissues.3.RT-PCR,Western blot and immunohistochemical analysis were performed to examine the expressions of Notch-1 and DLL4 in villus tissues.Results:1.Compared with induced abortion,the number of trophoblast cells was obviously decreased,accompanied by chromatic agglutination and karyopyknosis in the ESA villi.2.The mRNA levels of VEGF and VEGFR1 in ESA villi tissues were 1.42-fold and 1.85-fold lower than those in the respective induced abortion tissues(p<0.05).The protein levels of VEGF and VEGFR1 in ESA villi tissues were 2.27-fold and 1.59-fold lower than those in the respective induced abortion tissues(p<0.05).Meanwhile,the mRNA and protein levels of VEGFR2 in ESA villi tissues were 1.52-fold and 1.41-fold higher respectively than those in induced abortion tissues(p<0.05).Furthermore,we observed slightly stained VEGF,VEGFR1 in ESA villous trophoblast cells and VEGFR2 in induced aborted trophoblast cells.The dispersed stained VEGF,VEGFR1 in induced aborted trophoblast cells and VEGFR2 in ESA villous trophoblast cells were identified simultaneously,which was consistent with the results from RT-PCR and Western blot.3.Compared with induced abortion,Notch-1 was significantly downregulated and DLL4 was notably upregulated in ESA villus tissues.The expression of Notch-1 at protein and mRNA levels was declined significantly by 1.59-fold and 2.7-fold respectively in the villi of patients with ESA compared with patients with induced abortion(p<0.05).Meanwhile,the expression of DLL4 at protein and mRNA levels was increased notably by 1.91-fold and 2.3-fold respectively in the villi of patients with ESA compared with patients with induced abortion(p<0.01).Similarly,we detected dispersed expression of immunohistochemical Notch-1 and slight expression of immunohistochemical DLL4 in villous trophoblast cells from induced abortion patients.On the contrary,the faintly stained Notch-1 and prominently stained DLL4 were discovered in ESA villous trophoblast cells.Conclusions:1.The number of trophoblast cells was sharply reduced,and apoptotic number was increased in the ESA compared with induced abortion villi.2.The risk of ESA was raised along with the imbalanced VEGF/VEGFRs expressions in the villi.3.Notch-1 pathway was inhibited in trophoblast cells of spontaneous aborted villi.In summary,our data preliminarily revealed the pathological changes in villus tissues from patients with ESA,and addressed the expressions of Notch-1 pathway in ESA villus tissues.Therefore,we speculate that downregulated Notch-1 may trigger ESA through blocking the survival and angiogenesis of trophoblast cells.DecreasedNotch-1 level in trophoblast cells may serve as a reference index to assess ESA.Part two.Notch-1 pathway promotes the survival and angiogenesis of trophoblast cellsBackground and Objective:As our previous data indicated that downregulation of Notch-1 may induce cytostatic and anti-angiogenic effects of trophoblast cells,which is closely associated with ESA.Accumulating evidence suggests that Notch pathway could promote the proliferation,metastasis and angiogenesis in multiple malignancies,inhibiting Notch pathway by DAPT significantly enhances the apoptosis and represses the angiogenesis in various tumor cells.There are some similarities between the implantation process of gestational placenta and the development of tumor.In addition,downregulated Notch proteins were positively correlated with the reduction of placental weight in fetal growth retardation(FGR)placentas.In light of this,we further speculate that Notch pathway regulates the proliferation,apoptosis and angiogenesis in trophoblast cells.Hence,this study cultured trophoblast cells separated from induced abortion females in vitro,and inhibited Notch pathway by DAPT to explore the effect of Notch pathway on the proliferation,apoptosis and angiogenesis in trophoblast cells.Then we investigated the role of overexpressed Notch-1 in the apoptosis and angiogenesis with 50 μM DAPT treatment of trophoblast cells.Methods:1.10 cases of villus tissues from induced abortion females were recruited and the trophoblast cells were isolated and cultured.Immunofluorescence assay was employed to identify the expressions of cytokeratin and vimentin in cultured trophoblast cells.2.Trophoblast cells were treated with different concentrations of DAPT(0,25,50 μM)for 72 h for subsequent RT-PCR and Western blot experiments to detect the mRNA and protein levels of Notch-1 in trophoblast cells,3.Trophoblast cells were exposed to an increased concentration of DAPT(0,6.25,12.5,25,50,100 μM)for 24 h,48 h,and 72 h respectively for MTT assay to choose the most suitable concentration and time for other experiments and evaluate the effect of inhibiting Notch pathway on the proliferation of trophoblast cells.4.Trophoblast cells were subjected to various concentrations of DAPT(0,25,50μM)for 72 h,and flow cytometry was carried out to observe the effect of Notch pathway on apoptosis of trophoblast cells.The expression and distribution of Bcl-2,Bax and caspase-3 were measured by RT-PCR and immunofluorescence assay.5.Trophoblast cells were subjected to various concentrations of DAPT(0,25,50μM)for 72 h,then Western blot was conducted to analyze the expressions of VEGFR1 and VEGFR2.Furthermore,microvascular angiogenesis assay was applied to investigate the alteration of angiogenesis in trophoblast cells after inhibiting Notch pathway.6.Overexpressed intracellular NICD vector was constructed and transfected into trophoblast cells for RT-PCR detection of Notch-1 expression under 50 μM DAPT administration for 72 h.Then flow cytometry assay was carried out to investigate the effect of overexpressing Notch-1 on the apoptosis of trophoblast cells with 50 μM DAPT administration for 72 h7.Western blot and microvascular angiogenesis assay were performed to detect the effect of overexpressing Notch-1 on the expression of VEGFR1 and VEGFR2 and angiogenesis respectively in trophoblast cells upon50 μM DAPT administration for 72 h.Results:1.Trophoblast cells isolated from villous grew well,and the confluent cell monolayer appeared as cobblestone with keratin expression but without vimentin expression,which is consistent with epithelial cell characteristics,and suggests successfully cultured trophoblast cells in vitro.2.The expression of Notch-1 at mRNA and protein levels were both decreased significantly under 25μM and 50 μM DAPT treatment respectively compared with 0μM DAPT treatment group(p<0.001).3.The proliferation of trophoblast cells was inhibited notably upon 50μM and 100 μM DAPT treatments for 48 h compared with non-DAPT treatment cells(p<0.001),and cell viability exhibited significant dose-dependent inhibitions after an increased concentration of DAPT-treated for 72 h(p<0.001),indicating that inhibiting Notch pathway suppresses the proliferation of trophoblast cells.Moreover,the inhibitory rate was raised sharply from 25 μM to 50 μM DAPT treatment,so we chose these two concentrations for subsequent experiment.4.Flow cytometry results showed that DAPT enhanced the total apoptosis rate in a dose-dependent manner,suggesting that inhibiting Notch pathway boosts the apoptosis of trophoblast cells.Further RT-PCR and immunofluorescence assay confirmed the dramatically downregulated Bcl-2 and upregulated Bax in the cytoplasm of trophoblast cells treated with DAPT(p<0.001),at the same time,the expressions of caspase-3 was elevated apparently in DAPT-treated trophoblast cells compared with non-DAPT treated control(p<0.001).5.The expression of VEGFR1 was declined significantly after DAPT treatment(P<0.001)along with the remarkably elevated VEGFR2 expression compared with non-DAPT treatment trophoblast cells(p<0.001).Simultaneously,the microvascular angiogenesis of trophoblast cells was extremely attenuated upon DAPT administration.6.Both Hochest staining and flow cytometry results showed that overexpression of Notch-1 decreased the apoptosis rate of trophoblast cells upon 50 μM DAPT administration for 72 h(p<0.01).7.Compared with control,overexpression of Notch-1 upregulated VEGFR1 and downregulated VEGFR2,and further promoted the microvascular angiogenesis of trophoblast cells with 50 μM DAPT administration for 72 h.Conclusions:1.Inhibiting Notch pathway by DAPT suppresses the proliferation of trophoblast cells.2.Inhibiting Notch pathway by DAPT enhances the apoptosis of trophoblastcells by activating mitochondrial apoptotic pathway.3.Inhibiting Notch pathway by DAPT attenuates the angiogenesis of trophoblast cells by disturbing the balance of VEGF/VEGFRs expressions.4.Overexpressing Notch-1 could reverse the apoptosis of trophoblast cells induced by DAPT.5.Overexpressing Notch-1 could reverse angiogenic defects of trophoblast cells induced by DAPT.Taken together,our study proved that inhibiting Notch-1 pathway represses blastocyst implantation through suppressing the proliferation and angiogenesis and promoting the apoptosis of trophoblast cells,and preliminarily identified the mechanism on Notch-1-mediated ESA.Overall,upregulating Notch pathway may serve as a potential candidate in treatment of ESA.